シロイヌナズナ由来過酸化リン脂質グルタチオンペルオキシダーゼ様遺伝子のクローニングと発現

A cDNA encoding Arabidopsis purative phosphplipid hydroperoxide gultathione peroxidase (PHGPX) was cloned and sequenced by the reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends methods. The cDNA comprised 803 bp, and included an open reading frame which encodes a polypeptide of 169 amino acid residues with a molecular mass of 18,600 Da. The deduced amino acid sequence showed homology to plant putative PHGPXs and mammalian PHGPXs. The cloned gene was expressed in Escherichia coli cells to prouce an extra protein, which showed a molecular mass similar to the deduced one.シロイヌナズナから過酸化リン脂質グルタチオンペルオキシダーゼと高い相同性を持つタンパク質をコードするcDNAを単離し、その塩基配列を決定した。本遺伝子は、全長803bpからなり、169アミノ酸残基のタンパク質をコードしていた。アミノ酸配列は植物由来過酸化リン脂質グルタチオンペルオキシダーゼ様タンパク質と約80％の相同性を、哺乳類由来過酸化リン脂質グルタチオンペルオキシダーゼと約50％の相同性を示した。本遺伝子を大腸菌中で発現させた結果、遺伝子から予測される分子量をもつタンパク質が新たに生産された

Purification and Characterization of α-Glucosidases from Spinach Seeds

Four molecular forms of α-glucosidase were isolated from spinach seeds by several kinds of chromatography. The molecular masses of α-glucosidases Ⅰ,Ⅱ,Ⅲ,and Ⅳ were 78,78,82 and 82kDa by SDS-PAGE, and 62,62,190,and 70kDa by gel filtration, respectively. α-Glucosidases Ⅰand Ⅱ showed similar enzymatic properties. The Km for soluble starch was about 10 times lower than that for maltose, and they had higher activity not only towards malto-oligosaccharides but also towards α-glucans. The optimum pH was 4.5-5.5 and about 50% of the activity remained after incubation at 71℃ for 20 min. On the other hand, α-glucosidases Ⅲ and Ⅳ showed similar enzymatic propreties. The Km for maltose was 3-4 times lower than for solble starch, and they had high activity toward malto-oligosaccharides but faint activity towards α-glucnas. The optimum pH was 4.5-5.0 and no activity was found after incubation at 70℃ for 20 min. However, anti-α-glucosidase Ⅲ serum precipitated specifically with α-glucosidase Ⅲ.α－グルコシダーゼの分子多型が各種クロマトグラフィーによりホウレンソウ種子から単離された。α－グルコシダーゼⅠ、Ⅱ、Ⅲ、Ⅳの分子量は、それぞれSDS－PAGEにより78、78、82、82kDA、ゲル濾過のより62,62,190、70kDであった。α-グルコシダーゼⅠとⅡは、可溶性デンプンを基質とした際のKm値がマルトースよりも約10倍低く、さらに、α－グルカンだけでなくマルトオリゴ糖に対しても高い活性を示すことから、同じような酵素的特性を持つことが示された。最適pHは4.5ｶﾗ5.5を示し、温度安定性は70℃、20分処理で約50％の残存活性を示した。一方、α－グルコシダーゼⅢとⅣは、マルトースを基質とした際のKm値が可溶性デンプンよりも3－4倍低い値を示し、マルトオリゴ糖に対しては高い活性を示すが、α－グルカンに対してはほとんど活性を示さないことから、同じような酵素的特性を有することが示された。最適pHは4.5－5.5、そして70℃、20分処理では活性が認められなかった。しかしながら、抗α－グルコシダーゼⅢ血清は、α－グルコシダーゼⅢに対してのみ特異的に沈降線を形成した

Transcriptomic analysis of developing seeds in a wheat (Triticum aestivum L.) mutant RSD32 with reduced seed dormancy

Seed dormancy, a major factor regulating pre-harvest sprouting, can severely hinder wheat cultivation. Reduced Seed Dormancy 32 (RSD32), a wheat (Triticum aestivum L.) mutant with reduced seed dormancy, is derived from the pre-harvest sprouting tolerant cultivar, 'Norin61'. RSD32 is regulated by a single recessive gene and mutant phenotype expressed in a seed-specific manner. Gene expressions in embryos of 'Norin61' and RSD32 were compared using RNA sequencing (RNA-seq) analysis at different developmental stages of 20, 30, and 40 days after pollination (DAP). Numbers of up-regulated genes in RSD32 are equivalent in all developmental stages. However, down-regulated genes in RSD32 are more numerous on DAP20 and DAP30 than on DAP40. In central components affecting the circadian clock, homologues to the morning-expressed genes are expressed at lower levels in RSD32. However, higher expressions of homologues acting as evening-expressed genes are observed in RSD32. Homologues of Ca2+ signaling pathway related genes are specifically expressed on DAP20 in 'Norin61'. Lower expression is shown in RSD32. These results suggest that RSD32 mutation expresses on DAP20 and earlier seed developmental stages and suggest that circadian clock regulation and Ca2+ signaling pathway are involved in the regulation of wheat seed dormancy

ホウレンソウ種子に存在するα-グルコシダーゼの分子多型変化

Two molecular forms of α-glucosidase were isolated from spinach seeds after storage at 4℃ by CM-cellulose column chromatography and gel filtration. The molecular masses of α-glucosidase A and B were 78 kDa and 82 kDa by SDS-PAGE, and 62 kDa and 70 kDa by gel filtration, respectively. α-Glucosidase A had high activity not only toward maltooligosaccharides but also toward α-glucans. The optimum pH was 4.5-5.5 and about 50% of the activity remained after incubation at 65℃ for 20 min. On the other hand, α-glucosidase B had high activity toward maltooligosaccharides but faint activity toward α-glucans. The optimum pH was 5.0 and no activity was found after incubation at 65℃ for 20 min. The enzymatic and immunological properties of α-glucosidase A and B were similar to those of α-glucosidase. Ⅰor Ⅱ, and α-glucosidase Ⅲ or Ⅳ, isolated from spinach seeds without 4℃ storage, respectively. These findings suggest that the α-glucosidase in spinach seeds is modified to be two molecular forms.4℃保存したホウレンソウ種子から2種のα-グリコシダーゼをCM-セルロースカラムクロマトグラフィーとゲル濾過により精製した。α-グルコシダーゼAとBの分子量はそれぞれSDS-PAGEで78kDA、82kDa、ゲル濾過で62kDa、70kDaであった。α-グルコシダーゼAはマルトオリゴ糖だけではなく可溶性デンプンに対して強い加水分解活性を示した。至適pHは4.5-5.5であり、65℃、20分処理後に約50％の残存活性を示した。一方、α-グルコシダーゼBはマルトオリゴ等に対して強い加水分解活性を示したが、可溶性デンプンに対してほとんど活性を示さなかった。至適pHは5.0であり、65℃、20分処理後に活性を消失した。α-グルコシダーゼAとα-グルコシダーゼBは、4℃保存を行わない種子に見出されたα-グルコシダーゼⅠ、Ⅱとα-グルコシダーゼⅢ、Ⅳにそれぞれ酵素化学的性質、免疫化学的性質が類似していた。これらの結果、ホウレンソウ種子に存在するα-グルコシダーゼの分子多型は2種類に収束されることが示唆された

Rice Nudix Hydrolase OsNUDX2 Sanitizes Oxidized Nucleotides

Nudix hydrolase (NUDX) hydrolyzes 8-oxo-(d)GTP to reduce the levels of oxidized nucleotides in the cells. 8-oxo-(d)GTP produced by reactive oxygen species (ROS) is incorporated into DNA/RNA and mispaired with adenine, causing replicational and transcriptional errors. Here, we identified a rice OsNUDX2 gene, whose expression level was increased 15-fold under UV-C irradiation. The open reading frame of the OsNUDX2 gene, which encodes 776 amino acid residues, was cloned into Escherichia coli cells to produce the protein of 100 kDa. The recombinant protein hydrolyzed 8-oxo-dGTP, in addition to dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP), as did Arabidopsis AtNUDX1; whereas the amino acid sequence of OsNUDX2 had 18% identity with AtNUDX1. OsNUDX2 had 14% identity with barley HvNUDX12, which hydrolyzes 8-oxo-dGTP and diadenosine tetraphosphates. Suppression of the lacZ amber mutation caused by the incorporation of 8-oxo-GTP into mRNA was prevented to a significant degree when the OsNUDX2 gene was expressed in mutT-deficient E. coli cells. These results suggest that the different substrate specificity and identity among plant 8-oxo-dGTP-hydrolyzing NUDXs and OsNUDX2 reduces UV stress by sanitizing the oxidized nucleotides

Serine racemases from barley, Hordeum vulgare L., and other plant species represent a distinct eukaryotic group: gene cloning and recombinant protein characterization

Several D-amino acids have been identified in plants. However, the biosynthetic pathway to them is unclear. In this study, we cloned and sequenced a cDNA encoding a serine racemase from barley which contained an open reading frame encoding 337 amino acid residues. The deduced amino acid sequence showed significant identity to plant and mammalian serine racemases and contained conserved pyridoxal 5-phosphate (PLP)-binding lysine and PLP–interacting amino acid residues. The purified gene product catalyzed not only racemization of serine but also dehydration of serine to pyruvate. The enzyme requires PLP and divalent cations such as Ca2+, Mg2+, or Mn2+, but not ATP, whereas mammalian serine racemase activity is increased by ATP. In addition to the results regarding the effect of ATP on enzyme activity and the phylogenetic analysis of eukaryotic serine racemases, the antiserum against Arabidopsis serine racemase did not form a precipitate with barley and rice serine racemases. This suggests that plant serine racemases represent a distinct group in the eukaryotic serine racemase family and can be clustered into monocot and dicot types.</p

Singlet Oxygen Generation by Porphyrins and Metalloporphyrins Revisited: a Quantitative Structure-property Relationship (QSPR) Study

state followed by formation of singlet oxygen (1O2), which is a highly reactive species and mediates various oxidative processes. The design of advanced sensitizers based on porphyrin compounds have attracted significant attention in recent years. However, it is still difficult to predict the efficiency of singlet oxygen generation for a given structure. Our goal was to develop a quantitative structure-property relationship (QSPR) model for the fast virtual screening and prediction of singlet oxygen quantum yields for pophyrins and metalloporphyrins. We performed QSPR analysis of a dataset containing 32 compounds, including various porphyrins and their analogues (chlorins and bacteriochlorins). Quantum-chemical descriptors were calculated using Density Functional Theory (DFT), namely B3LYP and M062X functionals. Three different machine learning methods were used to develop QSPR models: random forest regression (RFR), support vector regression (SVR), and multiple linear regression (MLR). The optimal QSPR model «structure – singlet oxygen generation quantum yield» obtained using RFR method demonstrated high determination coefficient for the training set (R2 = 0.949) and the highest predicting ability for the test set (pred_R2 = 0.875). This proves that the developed QSPR method is realiable and can be directly applied in the studies of singlet oxygen generation both for free base porphyrins and their metal complexes. We believe that QSPR approach developed in this study can be useful for the search of new poprhyrin photosensitizers with enhanced singlet oxygen generation ability

Anti-Inflammatory Effect on Colitis and Modulation of Microbiota by Fermented Plant Extract Supplementation

Although results of recent studies suggest that fermented foods strongly affect the gut microbiota composition and that they relieve inflammatory bowel disease symptoms, some reports have described that fermented foods increase some inflammation markers based on differences in fermented food materials. This study evaluated the effects of fermented plant extract (FPE) on dextran sulfate sodium (DSS)-induced colitis in mice and the effects on fecal microbiota composition in humans. Mice fed 5% FPE with 3% DSS (FPE group) showed no body weight loss, atrophy of colonic length, or bloody stool, similar to mice fed a basal diet (negative group), whereas mice fed 3% DSS (positive group) exhibited those effects. Concentrations of inflammation markers IL-6 and TNF-alpha were not significantly different between FPE and negative groups; however, those concentrations became higher in the positive group. 16S ribosomal RNA gene sequencing was used to characterize fecal microbiota in healthy women before and after 3-month FPE supplementation. The FPE supplementation induced increases in Firmicutes phyla and in Clostridiales order, which play a central role in inflammation suppression. These results suggest that FPE enhances Clostridiales growth in the gut and that it has an anti-inflammatory effect

外反母祉における種子骨の偏位と関節症変化の関係

BACKGROUND: Although the tangential sesamoid view is used to visualize the sesamoid position relative to the first metatarsal head, correctly evaluating patients with severe varus of the first metatarsal is difficult. Computed tomography (CT) can be helpful due to its cross-sectional images in any plane. The purposes of this study were to evaluate the alignment of the tibial sesamoid and investigate the relationship between malalignment and degenerative change in the sesamoid metatarsal joint (SMJ) using simulated weight-bearing CT imaging in patients with hallux valgus. METHODS: In total, 269 feet from 142 patients with hallux valgus were included. The mean age was 63.7 years (range, 33-87 years). An anteroposterior weight-bearing radiograph was assessed for sesamoid position into 3 grades: grade 1, the tibial sesamoid was medial to the axis of the first metatarsal; grade 2, the tibial sesamoid was located below the first metatarsal axis; and grade 3, the tibial sesamoid was lateral to the first metatarsal axis. The hallux valgus and intermetatarsal angles (HVA and IMA, respectively) were measured. The lateral shift of the tibial sesamoid relative to the first metatarsal was classified into 3 grades on simulated weight-bearing CT classification: grade 1, tibial sesamoid was entirely medial to the intersesamoid ridge; grade 2, tibial sesamoid was subluxated laterally but located below the intersesamoid ridge; and grade 3, tibial sesamoid was located entirely lateral to the intersesamoid ridge. The differences of HVA and IMA in each grade were confirmed by using 1-way analysis of variance with Bonferroni post hoc corrections. Furthermore, multiple linear regression analysis was used to predict the degenerative change in the SMJ for age, sex, sesamoid position determined by CT or plain radiography, HVA, and IMA. The χ2 test was used for descriptive statistics to analyze the agreement between radiography or CT classifications of sesamoid position against degenerative change in the SMJ. RESULTS: Based on the radiographic classification of the tibial sesamoid position, 7 feet were classified as grade 1, 72 were grade 2, and 190 were grade 3, respectively. Based on the CT classification, 34 feet were classified as grade 1, 116 were grade 2, and 119 were grade 3. Degenerative change in SMJ progressed according to the sesamoid shift relative to the first metatarsal using either radiography or CT. In radiography, statistically significant differences were found except for the difference in HVA between grades 1 and 2. In addition, statistically significant differences were found between HVA and IMA, along with the grades in CT.In multiple linear regression, degenerative change was correlated with age and sesamoid position in CT and radiographic classifications. CONCLUSION: Our study showed that lateral shift of the tibial sesamoid increased in association with progression of the hallux valgus deformity. Furthermore, increasing lateral shift of the tibial sesamoid was associated with worsening degenerative change within the SMJ. LEVEL OF EVIDENCE: Level III, retrospective comparative study.博士（医学）・甲第657号・平成28年11月24日© Copyright 2016 American Orthopaedic Foot & Ankle SocietyThe definitive version is available at " http://dx.doi.org/10.1177/1071100716661827

ケイ酸/亜鉛置換ストロンチウムアパタイトコーティングによるβ-リン酸カルシウムの骨誘導能促進効果

Background: β-Tricalcium phosphate (β-TCP) is a popular synthetic bone graft substitute with excellent osteoconductive properties and bioabsorbability. However, its osteoinductive properties are inferior to those of autologous or allogeneic bone. Trace elements such as strontium (Sr), silica (Si), and zinc (Zn) have been reported to promote osteogenesis in materials. In this study, we aimed to determine whether a Si/Zn-substituted Sr apatite coating of β-TCP could enhance osteoinductive properties. Methods: The apatite-coated β-TCP disks were prepared using nanoparticle suspensions of silicate-substituted Sr apatite (SrSiP) or silicate- and Zn-co-substituted Sr apatite (SrZnSiP). Bone marrow mesenchymal cells (BMSCs) from rat femur were cultured and subsequently seeded at a density of 1.0 × 106/cm2 onto apatite-coated and non-coated β-TCP disks. In vitro, the β-TCP disks were then placed in osteogenic medium, and lactate dehydrogenase (LDH) activity was measured from supernatants after culture for 2 days. Additionally, after culture for 14 days, the mRNA expression of genes encoding osteocalcin (OC), alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP-2), and vascular endothelial growth factor (VEGF) was evaluated by qRT-PCR. In vivo, the β-TCP disks were transplanted subcutaneously into rats that were sacrificed after 4 weeks. Then, the harvested disks were evaluated biochemically (ALP activity, OC content, mRNA expression of OC, ALP, BMP-2, and VEGF measured by qRT-PCR), radiologically, and histologically. Results: Significantly higher mRNA expression of almost all evaluated osteogenic and angiogenic genes was observed in the SrZnSiP and SrSiP groups than in the non-coated group, with no significant cytotoxicity elicited by the apatite coating in vitro. Moreover, in vivo, the SrZnSiP and SrSiP groups showed significantly higher osteogenic and angiogenic gene expression and higher ALP activity and OC content than the non-coated group (P < 0.05). Radiological and histopathological findings revealed abundant bone formation in the apatite-coated group. Conclusions: Our findings indicate that apatite coating of β-TCP improves osteoinductive properties without inducing significant cytotoxicity.博士（医学）・甲第805号・令和3年12月21日© 2021. The Author(s).Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data